Derivatives of partially desulphated glycosaminoglycans endowed with antiangiogenic activity and devoid of anticoagulating effect

ABSTRACT

Partially desulfated glycosaminoglycan derivatives are described, particularly heparin, and more particularly formula (I) compounds 
     
       
         
         
             
             
         
       
     
     where the U, R and R 1  groups have the meanings indicated in the description. These glycosaminoglycan derivatives exhibit antiangiogenic activity and are devoid of anticoagulant activity.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 11/028,512filed Jan. 4, 2005, which is a continuation-in-part of U.S. applicationSer. No. 10/967,255 filed Oct. 19, 2004, which is a continuation of U.S.application Ser. No. 10/182,185 filed Jul. 25, 2002, now abandoned,which is a 371 of PCT/IT01/00034 filed Jan. 24, 2001 which claimsbenefit of priority of Italian application RM2000A000041 filed Jan. 25,2000, the entire contents of the aforementioned applications are herebyincorporated by reference into this application.

The invention described herein relates to partly desulfated 10glycosaminoglycan derivatives, particularly heparins, to processes fortheir preparation, to their use as active ingredients for thepreparation of medicaments with an antiangiogenic activity, particularlyfor the treatment of tumors, such as, for example, the metastatic forms,and to pharmaceutical compositions containing them.

BACKGROUND OF THE INVENTION

The first molecule possessing antiangiogenic activity was discovered incartilage by Henry Brem and Judah Folkman in 1975. Since that year morethan 300 new molecules capable of inhibiting angiogenesis have beendiscovered.

In the early eighties, with the discovery of interferon (α/β) as aninhibitor of tumor angiogenesis, clinical experimentation was initiatedbased on this therapeutic approach.

The media caused quite a stir, when on 3 Mar. 1998 the NewYork Timespublished the news that two molecules, angiostatin and endostatin,discovered in J. Folkman's laboratories at the Harvard Medical Schooland Children's Hospital in Boston, were yielding very encouragingresults in the struggle against cancer. The high degree of efficacy ofthese two molecules in inhibiting angiogenesis substantially boosted thesearch for new compounds. At present, there are about thirty moleculesendowed with anticancer activity which act via an antiangiogenicmechanism, which have entered into the clinical trials stage [PhasesI-III] and almost the same number of companies and institutions areinvolved.

In the United States alone it is estimated that there are approximately9 million patients who could benefit from antiangiogenic therapy. At thepresent time, at least 4,000 patients have been enrolled into clinicaltrials using this therapy without any particular unwanted effects beingregistered.

Within the framework of the general concept of angiogenesis we shoulddistinguish between vasculogenesis, that is to say the formation ofblood vessels during embryonic development and angiogenesis in thestrict sense of the term, meaning the formation of new blood vessels(capillaries) during the postnatal life starting from pre-existingvessels. The importance of angiogenesis for the growth of solid tumorsis amply documented. Over the past three decades it has been reportedthat tumor growth, as well as the formation of metastases, are strictlydependent on the development of new vessels capable of vascularising thetumor mass.

The inhibition of angiogenesis underlies the formation of necroticmasses within the tumor or the induction of apoptosis in tumor cells.

There are clear-cut differences between neovascularisation in normaltissue and that in tumor tissue. In the former, the vascular endotheliumrepresents a quiescent tissue with a very low mitotic index of itsconstituent cells (renewal time measured in hundreds of days), and thevascular network is regular, relatively uniform, and suitable foradequately oxygenating all the tissues, without any arteriovenousconnection. In tumortissue, on the other hand, stimulation of theproliferation of endothelial cells gives rise to a high mitotic index inthe latter (mean renewal time 5 days), the neovascularisation isdistinctly irregular with areas of occlusion, sometimes with closedendings, with arteriovenous contacts at some points, and, lastly, thebasal membrane presents gaps, which at some points leads to tissuehypoxia. These differences offer the opportunity of identifying drugswhich selectively block tumor neovascularisation.

In a tumor, the neovascularisation does not always coincide with aprecise stage in the tumor development; there are, in fact, cases inwhich angiogenysis begins even before the development of the tumor (forexample, carcinoma of the uterine cervix), others in which the twophases are coincident (for example, carcinoma of the bladder andbreast), and others in which angiogenesis begins after the neoplasm (forexample, melanoma and ovarian carcinoma; see, for example, “Manual ofMedical Oncology”, IV ed. (1991) G. Bonadonna et al. Antiangiogenictherapy presents numerous advantages compared to traditional standardchemotherapy (Cancer Research 1998, 58, 31408-16):

a) specificity: its target is a process, i.e. tumor neovascularisation;

b) bioavailability: its target is the endothelial cells, which can beeasily reached without the problems of traditional chemotherapy whichacts directly on the tumor cells;

c) chemoresistance: this is perhaps the most important advantage of thistherapy; in fact, since endothelial cells, unlike tumor cells, aregenetically stable, drug resistance phenomena are unlikely to occur;

d) metastatic spread: blockade of the neovascularisation limits thepropagation of the tumor cells to other parts of the body via thebloodstream;

e) apoptosis: blockade of the vascular network in the tumor reduces thesupply of oxygen and nutrients to the tumor cells; apoptosis is favoredin these conditions;

f) reduced systemic tOxicity: toxic effects, such as myelosuppression,gastrointestinal effects and temporary hair loss, which are almostinvariably present with traditional chemotherapy, are not observed withantiangiogenic therapy.

A number of molecular elements are known to be involved in tumorangiogenesis (Oncology 1997, 54, 177-84). Pro- and anti-angiogenicendogenous factors are known to be involved in the biological regulatorymechanism in the formation of new vessels.

Among the angiogenic stimulators we should mention: fibroblast growthfactors (FGF), vascular endothelial growth factor (VEGF), angiogenin,transforming growth factor-a, tumornecrosis factor (TNF-α),platelet-derived endothelial cell growth factor, transforming growthfactor-β, an in-vitro inhibitor, but an in-vivo stimulator, placentalgrowth factor, interleukin-8, hepatocyte growth factor, platelet-derivedgrowth factor, granulocyte colony-stimulating factors, proliferin, theprostaglandins (PGE₁, PGE₂), GM1-GT1b, substance P, the bradykinins, andnitric oxide.

In contrast, the angiogenesis inhibitors include: the soluble receptorof bFGF, the interferons (α, β, γ), angiostatin, thrombospondin1,prolactin (16 kDa terminal amino fragment), platelet factor 4 (PF4), thetissue metalloproteinase (TIMP) inhibitors, placental proliferinrelatedpeptide, glioma-derived angiogenesis inhibition factor, the angiostaticsteroids, cartilage-derived inhibitor (CDI), the heparinases, 5interleukin-12, plasminogen activator inhibitor, the retinoids,endostatin, angiopoietin-2, genistein, nitric oxide and GM3.

The integrins are a vast family of transmembrane proteins that mediatecell-to-cell and cell-to-extracellular matrix interactions. Allintegrins are capable of recognising a common peptide sequenceArg-Gly-Asp (“universal cell recognition site”), though every integrinpreferentially recognizes a different conformation of this tripeptide.The inhibition of specific subtypes of integrins can also be of greatinterest from the pharmacological standpoint for the development ofangiogenesis inhibitors.

The control of protein kinase-C (PK-C) may also allow regulation ofangiogenesis. There are, in fact, classic PK-C inhibitors capable ofcompletely or partially blocking angiogenesis.

Despite the enormous investments and the involvement of large numbers ofinstitutional and private research centers, the cancer problemworld-wide is still far from being definitively solved. Though theprognosis of cancer victims has improved, with survival rates risingover the past 30 years on average from 30 to 50%, and the genetic,cellular and biochemical mechanisms involved in the development of atumor are now well known, the possibility of defeating or at leastlimiting this type of pathology is still a problem of keenly feltconcern and many aspects are still unsolved, such as the likelihood ofrecurrence, complete remission and metastatic spread of the primarytumor.

Since the late seventies, when Folkman's observations began to beconfirmed by the international scientific community, hundreds ofmolecules endowed with antiangiogenic activity have been isolated fromnatural sources (plants, fungi, biological fluids) and synthesized inthe laboratory.

A number of drugs are already in Phase III, such as Marimastat (BritishBiotech.), l'AG3340 (Prinomast-Agouron), and Neovastat (Aeterna), all ofwhich act mainly at the pulmonary level (SNCL) with a mechanisminvolving interference with the metalloproteinases. Also in Phase IIIare RhuMad VEGF (this is an anti-VEGF antibody by Genetech) andinterferon α (commercial), which are active against solid tumors thanksto their interference with pro-angiogenic growth factors, or TNP-470(TAP Pharm.) which acts directly on the endothelial cells. Lastly, drugssuch as CAl (NCI) and IM862 (Cytran) are active as antiangiogenic agentsbut with a non-specific and poorly known mechanism.

These above-mentioned drugs may, in a few years, become part of theoncologist's therapeutic armamentarium, but there are other moleculeswhich have recently been inserted in Phase I/II clinical trials such asCombretastatin (OxiGene), methoxy-oestradiol and endostatin (EntreMed),which on the basis of preclinical studies are very promising. Companiessuch as Bristol Myers-Squibb (with BMS-275291), Novartis (withCGS27023A) or Parke-Davis (with Suramin) are involved in thistherapeutic strategy.

Heparin

Heparin is a heterogeneous mixture of naturally occurringpolysaccharides of various lengths and various degrees of sulphationwhich possesses anticoagulant activity and is secreted by the connectivetissue mastcells present in the liver (from which it was firstisolated), in the muscles, lungs, thymus and spleen.

In addition to the regular sequence, a sequence corresponding to theactive site for antithrombin activity has been identified in heparin.

The antitumorand antimetastatic activity of heparin and its derivativesis due to its ability to inhibit heparanase, to block growth factors andto regulate angiogenesis.

Heparan Sulphates (HS)

Heparan sulphates (HS) are ubiquitous protein ligands. The proteins bindto the HS chains for a variety of actions from simple immobilisation orprotection against the proteolytic degradation action to specificmodulations of biological activities, such as angiogenesis.

The carbohydrate skeleton, in both heparin and the heparin sulphates(HS), consists in an alternation of D-glucosamine (GlcN) and hexuronicacids (Glc.A or IdO.A).

In heparin, the GlcN residues are mainly N-sulphated, whereas in HS theyare both N-sulphated and N-acetylated, with a small amount ofunsubstituted N—.

HS is also on average less O-sulphated than heparin.

The use of heparin in the treatment of angiogenesis disorders, such astumors, particularly metastases, is substantially limited by theanticoagulant activity of heparin.

Chemical modifications have been made to heparin so as to reduce itsanticoagulant capacity, at the same time preserving itsantitumorproperties.

The opening of a unit of glucuronic acid in the antithrombin sitereduces the affinity of heparin for antithrombin: in this way, heparinscan be used with reduced anticoagulant effects, but still retainingantiangiogenic properties.

Heparanases

“Heparanases” are enzymes belonging to a family of endoglycosidases thathydrolyse the internal glycoside bonds of the chains of heparansulphates (HS) and heparin.

These endoglycosidases are involved in the proliferation of tumor cells,in metastases and in the neovascularisation of tumors. This suggeststhey may also be involved in tumor angiogenesis as a result of therelease, from the extracellular matrix, of growth factors bound toheparin, such as aFGF (also called FGF-1), bFGF (also called FGF-2) andVEGF.

These enzymes are biological targets for antiangiogenic activity. In thescientific literature there are a large number of structure/activitycorrelation studies (see, for example, Lapierre F. et al., Glycobiology,vol. 6, (3), 355-366, 1996). Though many aspects have still to beclarified, studies have been reported regarding the inhibition ofheparanases by heparin and its derivatives, using specific tests whichhave led to the emergence of considerations of a structural type whichmay serve as guides for obtaining new, more selective derivatives.

In the above mentioned work of Lapierre et al., heparin derivatives aredescribed obtained by 2-O desulfation or by “glycol split” (oxidationwith periodate and subsequent reduction with sodium borohydride). Thesederivatives, defined here as “2-O-desulfated heparin” and “RO-heparin”,respectively, have partly maintained the antiangiogenic activity ofheparin as assessed by means of the CAM test in the presence ofcorticosteroids, as reported in Table III (ibid. page 360).

Heparins and FGF

FGFs regulate multiple physiological processes such as cell growth anddifferentiation, but also functions involved in pathological processessuch as tumorangiogenesis.

FGFs are growth factors (a family of more than 10 polypeptides, of whichthe acid (FGF-1) and basic FGFs (FGF-2) are the ones which have beenmost studied, which require a polysaccharide cofactor, heparin or HS, tobind to the FGF receptor (FGFR) and activate it.

Though the precise mechanism whereby heparin and HS activate FGFs isunknown, it is known, however, that heparin/FGF/FGFR form a“trimolecular” or “ternary” complex.

One mechanism postulated is that heparin and HS induce so-calledsandwich dimerisation of FGF, and the latter, thus dimerised, forms astable complex with FGFR.

Antimetastatic Activity of Heparin Derivatives

The ability of a primary tumor to generate metastatic cells is perhapsthe main problem facing anticancer therapy.

Heparin derivatives with a substantial ability to block heparanase seemto be equally capable of inhibiting angiogenesis both in primary tumorsand in metastases.

In addition, the inhibition of heparanase reduces the migration abilityof tumor cells from the primary tumor to other organs.

The following table gives an example of structure/antimetastaticactivity correlation in the case of heparin:

% inhibition Heparin 97 N-succinyl-heparin 60 N-succinyl-RO-heparin 58Low MW heparin 86 Low MW N-succinyl-heparin 61 Very low MW heparin 83 MW= molecular weight

The data in this table suggest that very short fragments of heparin arestill endowed with good antimetastatic activity, while this activity isreduced when the amine group of the glucosamine is bound to succinicacid.

The structural requisites of heparin-like molecules that favor theangiogenesis-inhibiting action can be grouped in two categories on thebasis of the target one intends to block:

a) blockade of heparanase, an enzyme that hydrolyses the glycoside bondsof the heparan sulphates, releasing growth factors.

To this end the heparin-like compounds preferably comprise sequences ofat least eight monosaccharide units containingN-acetyl-glucosamine-glucuronic acid (or N-sulphated glucosamine (see,for example, D. Sandback-Pikas et al. J. Biol. Chem., 273, 18777-18780(1998) and references cited).

b) blockade of angiogenic growth factors (fibroblast type: FGF-1 andFGF-2; vascular endothelium type: VEGF; vascular permeability type:VPF).

To this end the heparin-like compounds preferably have sequences atleast five monosaccharide units long, containing 2-suplhated iduronicacid and glucosamine N, 6-sulphated (see, for example, M. Maccarana etal. J. Bill. Chem., 268, 23989-23905 (1993)).

In the literature small peptides (5-13 amino acids) with antiangiogenicactivity (U.S. Pat. No. 5,399,667 of the University of Washington) whichact by binding to a thrombospondin receptor, or longer peptides (50amino acids approx.).

Modified platelet factors are known—(EP 0 589 719, Lilly), capable ofinhibiting endothelial proliferation, with IC₅₀=7 nM.

Oligosaccharide fragments with antiangiogenic activity have also beenamply described: it has been found, in fact, that by varying thecarbohydrate sequence the interaction selectivity can be increased.

In addition, heparin can be used as a vehicle for substances which arethemselves antiangiogenic, such as some steroids, exploiting theaffinity of heparin for vascular endothelial cells; see, for example, WO93/18793 of the University of Texas and Imperial Cancer ResearchTechnology, where heparins are claimed with acid-labile linkers, such asadipic acid hydrazine, bound to cortisol. The antiangiogenic effect ofthe conjugated molecules is greater than that of the unconjugatedmolecules, even when administered simultaneously.

In Biochim. Biophys. Acta (1996), 1310, 86-96, heparins bound tosteroids (e.g. cortisol) are described with a hydrazone group in C-20that present greater antiangiogenic activity than the twounconconjugated molecules.

EP 0 246 654 by Daiichi Sc. describes sulphated polysaccharides withantiangiogenic activity with Phase II studies.

EP 0 394 971 by Pharmacia & Upjohn—Harvard Coll. Describeshexa-saccharides-heparin fragments—with low sulphation, capable ofinhibiting the growth of endothelial cells and angiogenesis stimulatedby (FGF-1).

EP 0 618 234 by Alfa Wasserman describes a method for preparingsemisynthetic glycosaminoglycans with a heparin or heparin structurebearing a nucleophilic group.

WO 95/05182 by Glycomed describes various sulphated oligosaccharideswith anticoagulant, antiangiogenic and anti-inflammatory activity.

U.S. Pat. No. 5,808,021 by Glycomed describes a method for preparingsubstantially non-depolymerised 2-0, 3-0 desulfated heparin with adesulfation percentage in positions 2- of the iduronic acid (1, 2-0) andin position 3 of the glucosamine unit (A, 3-0) ranging fromapproximately 99 to approximately 75% of the original percentage. Thismethod envisages desulfation conducted in the presence of a cation of abivalent metal, exemplified by calcium or copper, followed bylyophilisation of the product obtained. The desulfated heparins haveantiangiogenic activity.

The aim of the invention described herein is to find optimalglycosaminoglycan structures for generating antiangiogenic activitybased on heparanase inhibition and/or FGF growth factor inhibitionmechanisms. An additional aim of the invention described herein is toprovide a medicament with antiangiogenic activity which is essentiallydevoid of the typical side effects of heparin derivatives, such as, forexample, anticoagulant activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of the NMR spectroscopy of the compound ST1514 and ofa non-modified heparin.

SUMMARY OF THE INVENTION

It has now been found that on subjecting a glycosaminoglycan, such as aheparin-like glycosaminoglycan, heparin or modified heparin, containingglucosamine residues with different degrees of N-desulfation andoptional subsequent total or partial N-acetylation, to controlled2-O-desulfation treatment of the iduronic units up to a degree ofdesulfation not greater than 60% of the total uronic units, theangiogenic growth factor binding properties are maintained.Surprisingly, heparin 2-O-desulfated to not more than 60% of its totaluronic units is markedly antiangiogenic.

The desulfation conducted in the conditions described in the presentinvention also produces the formation of iduronic units with an oxyranicring in position 2, 3. The opening of the oxyranic ring in theconditions described in the present invention gives rise to L-iduronicor L-galacturonic units.

It is an object of the invention described herein a glycosaminoglycanderivative, particularly desulfated heparin, selectively partiallydesulfated with a desulfation degree not exceeding 60% of the totaluronic units; these sulfation gaps reduce the length of the regularsequences constituted by the succession of disaccharide trisulphateunits.

In one particular embodiment, the invention described herein refers to aformula (I) compound

where the U ring may have the following meanings:

X and X′, which can be the same or different, are an aldehyde group orthe —CH₂-D group, where D is hydroxyl or an amino acid, a peptide or aresidue of a carbohydrate or oligosaccharide;

R and R₁, which can be the same or different, are an SO₃ or acetylresidue;

n and m, which can be the same or different, may vary from 1 to 40; thesum of n+m ranges from 6 to 40; the m:n ratio ranges from 10:2 to 1:1.Preferably, m is greater than or equal to n. Preferably n ranges from 40to 60% of the sum m+n. The symbol

indicates that the units marked m and n are statistically, distributedalong the polysaccharide chain and are not necessarily in sequence.

The compounds which are the subject matter of the invention describedherein, have interesting antiangiogenic properties, and are thereforeuseful as active ingredients for the preparation of medicaments for thetreatment of pathologies based on abnormal angiogenesis, andparticularly for the treatment of tumors, even more particularly for thetreatment of metastases.

Advantageously, the compounds according to the invention presentreduced, if not non-existent anticoagulant properties, thus avoiding orreducing the side effects typical of the heparins. A further advantagestems from the fact that the compounds according to the invention can becharacterized with instrumental analytical techniques, such as NMRspectroscopy, thus allowing process control which is absolutelydesirable from the industrial point of view.

Also in the case of modified heparins, molecular weight (MW) has a veryimportant function when making angiogenesis inhibitors. It is wellknown, in fact, that a reduction in molecular weight (MW) to valuescorresponding to penta-saccharide units does not lead to a loss ofantiangiogenic activity. On the contrary, it has been established that,beyond a certain length, the heparin chains favor rather than inhibitdimerisation and thus activation of FGF.

DETAILED DESCRIPTION OF THE INVENTION

What is meant by desulfation degree is the percentage of non-sulphatediduronic acids in relation to total uronic acids originally present inthe starting heparin. One initial preferred range for the desulfationpercentage is from approximately 40 to approximately 60%. Among theformula (I) compounds the preferred compound is:

heparin partially 2-O-desulfated with a molecular weight (MW) of 11200,a polydispersion index D of 1.3, a sulfation degree of 1.99 (expressedas the S03-:COO— molar ratio), a percentage of modified uronic acidscompared to total uronic acids of approximately 50% (hereinafter alsocalled ST1514). Said compound is comprised in formula (I) where, amongthe other corresponding definitions, m:n=1:1 and the units marked m andn are distributed along the polysaccharide chain in a regular,alternating manner.

The compounds according to the invention described herein are preparedby means of a process comprising:

a) basic treatment at a temperature ranging from ambient temperature toapproximately 100° C., preferably from 50 to 70° C., and even morepreferably at approximately 65° C., which leads to the elimination of acontrolled percentage of sulphate groups in position 2 of the iduronicacid and to the formation of epoxide groups; and, if desired

b) opening of said epoxide ring at approximately pH 7, at a temperatureranging from approximately 50° C. to approximately 100° C., preferablyat approximately 70° C., to yield residues of galacturonic acid; or,alternatively

c) opening of said epoxide ring at a temperature ranging fromapproximately 0° C. to 30° C., preferably at approximately 25° C., toyield residues of iduronic acid; and, if desired

d) oxidation of the diols with sodium period ate, to yield the openingof the glycoside ring and the formation of two aldehyde groups permodified residue;

e) reduction of said aldehyde groups to primary alcohol and, if desired,transformation of the D group to a group other than hydroxyl, asenvisaged in the different meanings assigned in formula (I)

f) optional acid hydrolysis to obtain oligosaccharides corresponding tothe regular sequences, or alternatively

g) partial enzymatic hydrolysis with an enzyme selected from the groupconsisting of lyase, heparinase, heparitinase, or equivalent of productsobtained in step e) to yield oligosaccharides, preferably tetra- orocta-saccharides, with the non-reducing terminal residue consisting ofunsaturated iduronic acid, the reducing residue consisting of an Nsulphoglucosamine and containing at least one residue of open iduronicacid; or, alternatively

h) the compound obtained in step a), or the product obtained in step b)is treated by partial enzyme hydrolysis; and, if desired

i) subjection of the products obtained in one of steps a), b) and e) topartial 6-O-desulfation; or, alternatively,

j) subjection of the starting heparin partially or totally 6-desulfatedto steps a), b) and e).

According to the invention described herein, the preferred compound is:heparin partially 2-O-desulfated, obtainable by the process describedabove, where step a) is conducted for 45 min at 60° C. and step b) at70° C. at pH 7, and having a molecular weight (MW) of 11200, apolydispersion index D of 1.3, a sulfation degree of 1.99 (expressed asthe S0₃-:COO— molar ratio), percentage of modified uronic acids comparedto total uronic acids of approximately 50% (hereinafter also calledST1514);

The molecular weights are determined by HPLC-GPC (high performanceliquid chromatography−gel permeation chromatography). The sulfationdegree is determined by conductimetry and the percentage of modifieduronic acids by ¹³C-NMR.

MW is the molecular weight, and D is the polydispersion index expressedas MW/Mn.

According to the invention described herein, the starting products areglycosaminoglycans of various origins, preferably naturally occurringheparins. It is also possible to use chemically modified heparins with apercentage content of N,6 disulphate ranging from 0 to 100%. Startingfrom products with a different 6-O-sulphated glucosamine content, it ispossible to modulate the length of the regular sequences between oneopen iduronic acid and another. The glycosaminoglycans according to theinvention that present opening of the glycoside ring are conventionallycalled RO derivatives by those skilled in the field, meaning by thisthat the glycoside ring has been opened by means of an oxidation action,followed by a reduction (Reduction-Oxidation —RO). This opening of theglycoside ring is also conventionally called “glycol split”, so-calledbecause of the formation of the two primary hydroxy present on the openring. The compounds referred to herein will also be called “RO”derivatives or “Glycol Split”.

In a further embodiment of the invention described herein, the aldehydesand primary hydroxy derived from the opening reaction described above(“glycol split”) also lend themselves to the subsequentfunctionalization. Therefore, formula (I) compounds may also bear equalor different groups, as defined above for X and X′, on the primaryhydroxy deriving from glycol split. for example, oligosaccharide orpeptide groups, ranging from a single saccharide or amino acid to morethan one unit of length, preferably 2 or 3 units.

Formula (I) compounds where X and X′ are —CH₂OH can also be used asvehicles for other types of drugs, by means of suitable binding with theheparin portion which is capable of providing a stable bond in normalconditions of manufacture and storage of a formulated drug which,however, releases the transported drug in the body, preferably in thevicinity of the target organ. Examples of drugs that can be transportedare steroidal and non-steroidal anti-inflammatory drugs,corticosteroids, and other drugs with an antimetastatic action, in whichcase there will be an advantageous enhancement of the antimetastaticeffect as a result of the sum of the separate intrinsic activities ofthe compounds according to the invention and the antimetastatic agentbound thereto, with the related advantages of a greater targetselectivity and lower systemic toxicity. Examples of these drugs are themetalloproteinase inhibitors. Other drugs which can be usefullytransported are those that act at the endothelial level. Formula (I)compounds where X and X′ are other than hydroxy or aldehyde can also beused as vehicles for drugs, in which case the X and X′ groups will actas “spacers” between the transported molecule, that is to say theglycosaminoglycan of the present invention and the molecule acting asthe vehicle, in those cases where this may be desirable for reasons ofpharmacokinetics or pharmacodynamics.

In the case of compounds according to the invention deriving fromheparin, these are prepared starting from heparin itself by means ofdesulfation techniques known to the technical experts in the field. Forexample, the desulfation is conducted in the presence of alkalineagents, such as sodium hydroxide, at temperatures ranging from ambienttemperature to 100° C., preferably from 50 to 70° C., for example at 60°C., for a sufficiently long period to obtain the desired desulfation.The desulfation is controlled by acting on the process parameters, suchas the concentrations of reactants, the temperature and the reactiontimes. One preferred example consists in maintaining constantconcentrations of substrate (glycosaminoglycan) at 80 mg/ml and of NaOHat 1 M, a constant temperature of 60° C. and controlling the desulfationwith a reaction time from 15 to 60 min. The expert in the field may varythe conditions, for example by raising the reaction emperature andshortening the reaction time, on the basis of normal trial and error inexperimental practice and on the basis of his or her general knowledgeof the subject.

The treatment with alkaline agents gives rise to an intermediate productcharacterized by the presence of an epoxide ring on the desulfated unit.In a thoroughly surprising manner, these intermediates have proved to beendowed with antiangiogenic properties similar to those of the formula(I) compounds. Therefore, a further object of the invention describedherein is a derivative of partially desulfated heparin, and thereforeheparin with a reduced charge, particularly heparin not de sulfated morethan 60%, characterized by an epoxide ring on the desulfation site. Saidcompounds characterized by an epoxide ring also belong to the objectscovered by the present invention, that is to say the pharmaceuticalcompositions that contain them and their use for the preparation ofmedicaments with antiangiogenic activity.

The following compounds are preferred:

heparin partially 2-O-desulfated with a molecular weight (MW) of 12900D, a polydispersion index D of 1.5, a sulfation degree of 2.05(expressed as the S0₃-:COO— molar ratio), percentage of modified uronicacids compared to total uronic acids: 5% epoxide groups, 29% oxidatedand reduced uronic residues (hereinafter also called ST1513);

heparin partially 2-O-desulfated with a molecular weight (MW) of 11000D, a polydispersion index D of 1.5, a sulfation degree of 2.05(expressed as the S0₃-:COO— molar ratio), percentage of modified uronicacids compared to total uronic acids: 5% epoxide groups, 29% uronicresidues oxidated and reduced uronic residues;

heparin partially 2-O-desulfated with a molecular weight (MW) of 9200 D,a polydispersion index D of 1.5, percentage of modified uronic acidscompared to total uronic acids: 11% epoxide groups, 27.5% oxidated andreduced uronic residues.

In one of the possible embodiments of the invention described herein,the following are preferred:

heparin partially 2-O-desulfated, obtainable by the process describedabove, where step a) is conducted for 15 min at 60° C. and step b) at70° C. at pH 7, and with a molecular weight of 12900 D, a polydispersionindex D of 1.5, a sulfation degree of 2.05 (expressed as the S0₃-:COO—molar ratio), percentage of modified uronic acids compared to totaluronic acids: 5% epoxide groups, 29% oxidated and reduced uronicresidues (hereinafter called ST1513);

heparin partially 2-O-desulfated, obtainable by the process describedabove, where step a) is conducted for 30 min at 60° C. and step b) at70° C. at pH 7 (hereinafter called ST1516), and with a molecular weight(MW) of 11000 D, a polydispersion index D of 1.5, a sulfation degree of1.8 (expressed as the S0₃-:COO— molar ratio), percentage of modifieduronic acids compared to total uronic acids. 5% epoxide groups, 29%oxidated and reduced uronic residues;

heparin partially 2-O-desulfated, obtainable by the process describedabove, where step a) is conducted for 60 min at 60° C. and step b) at70° C. at pH 7, and with a molecular weight (MW) of 9200 D, apolydispersion index D of 1.5, percentage of modified uronic acidscompared to total uronic acids: 11% epoxide groups, 27.50/0 oxidated andreduced (split) uronic residues (hereinafter called STI515).

Subsequent to the formation of the epoxide ring, the latter is opened,again resorting to known techniques. The percentage of epoxide formed iscalculated from the ratio between the areas of the ¹³C-NMR signals atapproximately 55 ppm, characteristic of carbons 2 and 3 of the uronicacid ring containing the epoxide and the total number of anomericsignals (C1 of the glucosamine and uronic acid residues). If the openingis conducted hot, a galacturonic acid residue is obtained, whereas, ifthe opening of the epoxide ring is conducted cold, an iduronic acidresidue is obtained. Preferred examples of compounds containing anepoxide ring are those obtainable by the process described above andhaving epoxidated uronic acid contents of 14% (hereinafter STI509), 24%(hereinafter STI525) and 30% (hereinafter STI526), respectively.

The partially desulfated heparin is then subjected to “glycol-split” (ROfor short), according to the process defined above and Smith degradation(SD for short).

Alternatively, formula (I) compounds can also be obtained withoutpassing via the epoxide intermediate, that is to say by direct glycolsplit and subsequent Smith degradation.

The process described so far leads to formula (I) compounds in which theX and X′ groups are both —CH₂OH.

For X and X′ other than —CH₂OH, methods are available to the expert inthe field for transforming the hydroxyl group with other groupsenvisaged in the definitions given above. For example, the conjugationwith amino acids or peptides can be done by treating the intermediatealdehyde derived from the glycol-split reaction with a reductiveamination reaction (Hoffmann J. et al. Carbohydrate Research, 117,328-331 (1983)), which can be conducted in aqueous solvent and iscompatible with maintenance of the heparin structure.

If desired, and this constitutes a further object of the inventiondescribed herein, the formula (I) compounds can be further degraded withacid agents in suitable pH conditions, e.g. at pH 4, to yield a mixtureof oligo saccharides that maintain the antiangiogenic properties.

In the same way, objects of the present invention are the compoundsobtained by one of the steps g), h), i) and j) of the process describedabove.

Objects of the invention described herein are pharmaceuticalcompositions containing as their active ingredient at least one formula(I) compound, alone or in combination with one or more formula (I)compounds, or, said formula (I) compound or compounds in combinationwith the desulfated heparins described above, e.g. the epoxidatedintermediates; the latter can also be used alone as active ingredientsin the pharmaceutical compositions. The active ingredient according tothe present invention will be in a mixture with suitable vehicles and/orexcipients commonly used in pharmaceutical technology, such as, forinstance, those described in “Remington's 10 Pharmaceutical SciencesHandbook”, latest edition. The compositions according to the presentinvention will contain a therapeutically effective quantity of theactive ingredient. The doses will be determined by the expert in thefield, e.g. the clinician or primary care physician according to thetype of disease to be treated and the patient's condition, orconcomitantly with the administration of other active ingredients. Byway of an example, doses ranging from 0.1 to 100 mg/kg may be indicated.

Examples of pharmaceutical compositions are those that can beadministered orally or parenterally, intravenously, intramuscularly,subcutaneously, transdermally or in the form of nasal or oral sprays.Pharmaceutical compositions suitable for the purpose are tablets, hardor soft capsules, powders, solutions, suspensions, syrups, and solidforms for extemporary liquid preparations. Compositions for parenteraladministration are, for example, all the intramuscular, intravenous andsubcutaneous injectable forms as well as solutions, suspensions andemulsions. Liposome formulations should also be mentioned. The tabletsalso include forms for the controlled release of the active ingredientwhether as oral administration forms, tablets coated with suitablelayers, microencapsulated powders, complexes with cyclodextrins, depotforms, for example, subcutaneous forms, such as depot injections orimplants.

The compounds according to the invention described herein possessantiangiogenic activity. This makes them suitable for the preparation ofmedicaments useful for the treatment of objects, generally mammals, andparticularly human subjects, suffering from altered angiogenesis.Examples of diseases treated with the medicament which is the object ofthe present invention are primary tumors, metastases, diabeticretinopathies, psoriasis, retrolenticular fibroplasia, restenosis afterangioplasty and coronary by-pass.

Advantageously, the compounds according to the present invention aresubstantially devoid of the side effects typical of heparin. Inparticular, the compounds according to the invention are substantiallydevoid of anticoagulant activity. By substantially devoid of suchactivity the expert in the field means no or only negligible activityfrom the point of view of clinical use.

One of the first growth factors found to have an angiogenic role wasbFGF (or FGF-2) followed shortly afterwards by aFGF (or FGF-1) (for areview of the subject see Christofori, Oxford University, 1996). Bothproteins are members of a class of growth factors characterized by ahigh degree of affinity for heparin. Other potent angiogenic inductorsare VEGF, VEGF-B and VEGF-C. All three VEGF factors, like the two FGFs,are expressed ubiquitously in the body. Both of these types of factors,a/bFGF (FGF-1 and FGF-2) and VEGF, bind to specific high-affinityreceptors with a transmembrane domain possessing tyrosine-kinaseactivity. The three receptors for the VEGFs-VEGF-1 (flt-1), VEGF-2(kdr/flk-1) and VEGF-3 (flt-4) are expressed specifically on endothelialcells, whereas the four FGF receptors are expressed in numerous organsand tissues (for a review of the subject see Risau and Flame 1995 Annu.Rev. Cell Dev: Biol. 11: 73-91). bFGF binding to heparin or to fragmentsof heparan sulphate causes their dimerisation and the possibility ofbinding to their own receptor by activating the transduction pathway ofthe signal that activates the endothelial cell in both mitogenesis anddifferentiation.

The inhibition of FGF binding to heparin or to fragments of heparansulphate thus represents a valid therapeutic target in combating thepathological neoangiogenesis induced by an increased local or systemiclevel of growth factors.

To this end an in-vitro model was designed capable of assessing theinterference of the various different heparin derivatives with bFGFbinding to its own receptor.

In particular, ovarian cells of Chinese hamsters (CHO) were used whichpossess heparan sulphates on their surface, but lack FGF receptor 1 forthe FGF called CHO—K1. CHO cells called CHO-745flg were also used, whichexpress the FGF receptor 1 but which, unlike the former group, do notpossess membrane heparan sulphates. These latter cells were stablytransfected with cDNA for green fluorescent protein and for this reasoncould be observed under the fluorescence microscope with a combinationof filters for the detection of green emission. In brief, the techniqueis based on the possibility that the two types of cells may interact(form stable bonds) only if FGF is added to the two cell types. In fact,in this case, the heparan sulphates of the CHO-745flg cells will bind toFGF, which in turn will bind to the CHO-K1 cells, establishing a bridge.The binding that occurs is detectable as a result of the greenfluorescence emitted by the CHO-745flg cells.

In addition, the compounds were assayed for their cell proliferationinhibiting activity. In fact, one of the main effects of FGF is tostimulate cell growth in the absence of serum in cells expressing FGFreceptor 1 (FGFR-1).

To this end, two cell lines were used, both expressing FGFR-1 (L6WT1 andbovine aorta cells), the growth of which was evaluated in the presenceof FGF with or without the addition of heparin derivatives for which aninhibitory activity was expected.

Inhibition of FGF2-Mediated Intercellular Adhesion

CHO-K1 cells were seeded at a density of 90,000 cells/cm2 in 24-wellplates. After 24 hours the cells were fixed in 3% glutaraldehyde in PBSfor 2 hours at 4° C. and washed with 0.1 M glycine/PBS. On the fixedmonolayers CHO 745/flg cells, transfected with FGFR-1, at a density of50,000 cells/cm2 in DMEM containing 10 mM EDTA, 30 ng/ml FGF-2 andincreasing doses of the test compounds. After 2 hours' incubation at 37°C., the adhering cells were counted under the inverted microscope. Theresults were expressed as percentage of the number of cells adheringcompared to those measured in the absence of the test compounds. Thecompounds were examined in triplicate at 6 concentrations ranging from 1ng/ml to 100 μg/ml and the ID₅₀ was calculated for each compound (Table1).

TABLE 1 Inhibition of intercellular adhesion (ID50) of CHO-Kl e CHO-745flg cells Product Inhibition (ID₅₀) Heparin 100 RO heparin 8 ST151380 ST1516 110 ST1514 105 ST1515 120 ST1525 50 ST1528 75 ST1507 125

Inhibition of DNA Synthesis in L6 Cells Transfected with FGFR-1

L6-WT1 cells (rat myoblasts transfected with FGFR-1) were seeded at adensity of 25,000 cells/cm² in 48-well plates in DMEM+10% FCS. After 24hours the cells were washed with serum-free medium and incubated for 48hours with DMEM+0.5% FCS. The cells were then incubated for 16 hourswith FGF-2 at the concentrations of 15 and 30 ng/ml in the presence orabsence of the test compounds (all at 100 μg/ml). At the end of theincubation 3H-thymidine (0.25 μCi/well) was added without changing themedium. After 6 hours, precipitable TCA was measured. Each experimentalpoint is the mean of 3 determinations. The results are given in Table 2.

TABLE 2 Inhibition of DNA synthesis in L6 cells transfected with FGFR-1.Incorporation of 3H-thymidine Product (% vs. control) Heparin (FGF-2 15ng/ml) 50 Heparin (FGF-2 30 ng/ml) 74 Heparin RO (FGF-2 15 ng/ml) 41Heparin RO (FGF-2 30 ng/ml) 80 ST1513 (FGF-2 15 ng/ml) 93 ST1513 (FGF-230 ng/ml) 102 G3025B (FGF-2 15 ng/ml) 68 G3025B (FGF-2 30 ng/ml) 71ST1514 (FGF-2 15 ng/ml) 58 ST1514 (FGF-2 30 ng/ml) 99 ST1515 (FGF-2 15ng/ml) 79 ST1515 (FGF-2 30 ng/ml) 100

Effect on DNA Synthesis in Bovine Aorta Endothelial Cells (BAEC)

BAEC cells were seeded at a density of 2,500 cells/cm² in 48-well platesin EGM Bullet Kit complete medium. After 24 hours the cells werecultivated in the absence of serum in EGM medium without bovine brainextract and hEGF. After a further 24 hours, the cells were treated with30 ng/ml of bFGF in the presence of increasing concentrations of thetest compound. After 16 hours, 1 μCi/ml of 3H-thymidine was added to themedium. After 6 hours, precipitable TCA activity was measured. Eachexperimental point is the mean of 8 determinations. The results aregiven in Tables 3-6.

TABLE 3 Effect on DNA synthesis in bovine aorta endothelial cells(BAEC) - ST1514 Incorporation of Concentration ³H-thymidine ng/ml (% vscontrol) 1 80 10 78 100 42 1000 33 10000 22 100000 5

TABLE 4 Effect on DNA synthesis in bovine aorta endothelial cells(BAEC) - ST1528 Incorporation of Concentration ³H-thymidine ng/ml (% vscontrol) 1 85 10 73 100 85 1000 32 10000 28 100000 5

TABLE 5 Effect on DNA synthesis in bovine aorta endothelial cells(BAEC) - ST1525 Incorporation of Concentration ³H-thymidine ng/ml (% vscontrol) 1 75 10 67 100 26 1000 21 10000 7 100000 2

TABLE 6 Effect on DNA synthesis in bovine aorta endothelial cells(BAEC) - ST1507 Incorporation of Concentration ³H-thymidine ng/ml (% vscontrol) 1 92 10 92 100 45 1000 40 10000 18 100000 12

Test of Cell Proliferation in BAEC

BAEC cells at the 7th pass were seeded at a density of 2,500 cells/cm²in 96-well plates in EBM medium without bovine brain extract and hEGF.To this medium were added 30 ng/ml of FGF-2 and each of the testcompounds at 5 concentrations ranging from 10 ng/ml to 100 μ/ml. After 3days the cells were fixed and stained with crystalviolet and the opticaldensity was determined by means of an ELISA microplate reader. Eachexperimental point was done in quadruplicate and the ID50 wascalculated. The results are given in Table 7.

TABLE 7 Test of cell proliferation in BAEC Inhibition (ID₅₀) Productμg/ml Heparin 100 ST1509 0.02 ST1525 10 ST1526 0.02 ST1527 100 ST15280.1

Test of Cell Proliferation in Foetal BAEC GM 7373

GM7373 cells were seeded at a density of 70,000 cells/cm² in 96-wellplates in MEM+10% FCS medium. After 24 hours, the cells were washed withserum-free medium and treated with 10 ng/ml of FGF-2 in mediumcontaining 0.4% FCS. After 8 hours, the test compounds were added to themedium at 5 concentrations ranging from 10 ng/ml to 100 μg/ml. After afurther 16 hours, the cells were trypsinised and counted in a Burkerchamber. The ID₅₀ was calculated for each compound and the results arethe means of 2 experiments in triplicate. The results are given in Table8.

TABLE 8 Test of cell proliferation in foetal BAEC GM 7373 Inhibition(ID₅₀) Product μg/ml Heparin 2 ST1509 0.3 ST1525 2 ST1526 1 ST1527 20ST1528 0.2 ST1514 0.1

Inhibition of Angiogenesis

For the study of angiogenesis the model used was the chick embryochorioallantoic membrane model (Ribatti e al. Int. J. Dev. Biol., 40,1189 (1996)).

300 embryonated hens' eggs were incubated at 37° C. in constant humidityconditions. On the 3rd day of incubation, after aspirating 2-3 ml ofalbumen from the acute pole of the egg so as to detach the CAM from theshell, a window was cut with scissors in the shell. The vesselsunderlying the CAM were thus exposed. The window was closed with atransparent glass panel and the eggs were put back into the incubator.On the 8th day of incubation, in sterile conditions, the CAMs weretreated according to the protocol described below using a techniquedeveloped recently (Ribatti et al. J. Vasco Res. 34, 455 (1997)), whichinvolves the use of sterile gelatin sponges measuring 1 mm2 in size.Each molecule was resuspended in 3 μl of PBS at a concentration of 50 or100 μg/embryo. As a positive control, FGF-2 was used (1 μg/sponge), forwhich potent angiogenic activity on the CAM has been demonstrated(Ribatti et al., Dev. Biol. 170, 39, (1995)). The sponges were firstrested on the surface of the CAM, and then 3 μl of solution of the testsubstance were pipetted onto the surface of the sponge. The CAMs wereexamined daily using a Zeiss SR stereomicroscope equipped with aphotographic device. The experiments were discontinued on the 12th dayof incubation, when an overall assessment of the angiogenic activity ofthe molecules was carried out, expressed as percentage inhibition inrelation to the number of experiments initiated and completed (Table 9).In addition, for compound ST1514, assayed at the concentration of 100μg/embryo, a macroscopic quantitative evaluation of the angiostaticactivity was also carried out according to the technique proposed byBrooks et al. (Science, 264, 569, (1994)), counting the number ofvessels surrounding the sponge. The number of vessels was compared withthat of the control sponges treated with PBS (vehicle of the testcompound) and with FGF-2 (positive control). The results are given inTable 10.

TABLE 9 Angiostatic activity on CAM model Concentration N. InhibitionCompound (μg/embryo) experiments (%) N-acetylated 50 10 0 heparin ROheparin (ox- 50 10 20 red 19.6%) ST1514 (RO 56%) 50 10 50 ST1514 (RO56%) 100 10 80

It should be noted that naturally occurring heparin has no activity.

TABLE 10 Angiostatic activity: macroscopic quantitative evaluation(Brooks) N. vessels at sponge/CAM Compound N. embryos interface STI5I4(100 μg/embryo) 10 2 + 1 PBS (3 μl) 5 7 + 2 FGF-2 (1 μg/embryo) 5 50 +4 

It should be noted that naturally occurring heparin has no activity.

Evaluation of Heparin Toxicity in Balb/c Mice.

20 6-week-old female Balb/c mice weighing 20 g (Harlan), divided intogroups by casual randomisation, were treated with heparin sodium, as areference, and with the compound according to the present inventioncalled STI514. The treatment schedule was of the q2dx5 type, i.e. 5total administrations at intervals of 2 days, administering 200 μl/mouseof 50 mg/kg/10 ml and 25 mg/kg/10 ml solutions subcutaneously.

The substances were solubilized as follows:

Heparin sodium: the solution is prepared by solubilizing 160 mg ofpowder in 4 ml of PBS 1× pH 7.4 free of Ca⁺⁺- and Mg⁺⁺-ions; it issubdivided into aliquots of 243 μl and stored at −20° C. At the time oftreatment, the solution is diluted in Ca⁺⁺- and Mg⁺⁺-free PBS (Dulbecco,modified formula) 1×, pH 7.4 so as to have the substance at finalconcentrations of 50 mg/kg/10 ml and 25 mg/kg/10 ml.

ST1514: the solution is prepared by solubilizing 160 mg of powder in 4ml of Ca⁺⁺- and Mg⁺⁺-free PBS 1×, pH 7.4; it is subdivided into aliquotsof 243 μl and stored at −20° C. At the time of treatmen, the solution isdiluted in Ca⁺⁺- and Mg⁺⁺-free PBS (Dulbecco, modified formula) 1×, pH7.4 so as to have the substance at final concentrations of 50 mg/kg/10ml and 25 mg/kg/10 ml.

48 hours after the last administration of the test substances a bloodsample is taken for full blood count and haematological analysis.

Blood sampling: the mice are placed in a hermetically sealed box, intowhich is insufflated C0₂ in an amount sufficient to stun the animal.Blood is then taken from the retro-orbital plexus of each animal(approximately 1 ml of blood/mouse) which is distributed in amounts of0.4 ml of blood/mouse in Eppendorf test tubes containing 20 μl of Visterheparin (5000 U/ml) for carrying out the full blood count andhaematological analysis; and the same amount of blood is distributed inother Eppendorf test tubes containing 50 μl of 3.8% sodium citrate inorder to perform prothrombin time determinations. After taking the bloodsample, each animal is sacrificed by cervical dislocation.

Number of samples: 2 blood samples/mouse are taken, with which two fullblood counts are carried out and two slides and a plasma sample areproduced to be stored at −20° C.

Full blood count: the appliance (Cell Analyzer 580 A, DELCON) is usedaccording to the standard procedure described in the operating manual.The blood sample (25 μl) is taken by the dilutor and brought up to avolume of 10 ml (dil. 1:400) with isotonic solution (PLTA saline,DELCON). From this solution (called solution A), the dilutorautomatically takes a sample of 100 μl and brings it up to a volume of10 ml (dil. 1:100), thus obtaining solution B. To solution A are added 3drops of Emosol (DELCON) for lysis of the red blood cells. This solutionis used for the WBC reading. Solution B, on the other hand, is used forthe RBC and platelet (PLT) readings. Each sample is read in duplicate.

The data obtained will be analyzed using ANOVA.

The groups of animals treated with heparin sodium at the doses of 50mg/kg/10 ml and 25 mg/kg/10 ml present a marked haematoma at theinoculum site; this phenomenon does not occur in the groups submitted totreatment with ST1514. Autopsies conducted in the study animals revealthat the groups submitted to treatment with heparin sodium at doses of50 mg/kg/10 ml and 25 mg/kg/10 ml present livers with abnormalcharacteristics, whereas no such phenomenon is detected in the groupstreated with ST1514.

The data relating to the haematological analysis show a reduction in redblood cells in both groups treated with heparin sodium at the doses of50 mg/kg and 25 mg/kg as compared to the control group, whereas no suchdifference is detectable in the groups treated with ST1514.

The group treated with heparin sodium at the dose of 25 mg/kg presents asignificant platelet deficiency compared to the control group. None ofthe study treatment groups presents significant differences in thenumbers of white blood cells as compared to the control group.

The following examples further illustrate the invention.

Example 1

1 g of heparin is dissolved in 12.5 ml of NaOH 1N. The solution isheated and stirred at 60° C. for 45 minutes. The reaction is blocked byrapid cooling and neutralization. The solution is then heated at 70° C.at pH 7 until the epoxide ring is completely open (the reaction trend is20 controlled by NMR). The desulfated sample (here called G2999H) isdissolved in 20 ml of water and cooled to 4° C. After the addition of 20ml of a solution of NaIO₄ 0.2 M, the solution is left to stir in thedark for 20 hours, and the reaction is stopped by adding ethylene glycoland 48 the salts are eliminated by tangential ultrafiltration. 400 mg ofNaBH₄, subdivided into several portions, are added to the desaltedsolution (30-40 ml). The solution is left to stir for 3 hours at ambienttemperature, then neutralized with diluted HCl and desalted bytangential ultrafiltration. 710 mg of product, here called ST1514, areobtained.

Molecular weight (MW): 11200 D, polydispersion index D 1.3, sulfationdegree 1.9 (expressed as S0₃-:COO— molar ratio); the percentage ofmodified uronic acids compared to total uronic acids is approximately50%. The compound was characterized by means of NMR spectroscopy (FIG.1).

Examples 2-4

Adopting the same procedure as in example 1, with the exception that thebasic solution was heated for 15, 30 and 60 minutes, respectively,compounds with the following characteristics were obtained:

ST1513: molecular weight (MW) 12900 D, polydispersion index D 1.5,sulfation degree 2.05 (expressed as S0₃-:COO— molar ratio), percentageof modified uronic acids compared to total uronic acids: 5% epoxidegroups, 29% oxidated and reduced (split) uronic residues;

ST1516: molecular weight (MW) 12900 D, polydispersion index D 1.5,sulfation degree 1.8 (expressed as S0₃-:COO— molar ratio), percentage ofmodified uronic acids compared to total uronic acids: 5% epoxide groups,29% oxidated and reduced (split) uronic residues;

ST1515: molecular weight (MW) 9200 D, polydispersion index D 1.5,percentage of modified uronic acids compared to total uronic acids: 11%epoxide groups, 27.5% oxidated and reduced (split) uronic residues.

Example 5 Glycol-split N-acetyl beparins (NA-Hgs)

Fully N-acetylated heparin (¹⁰⁰NA-H,) was prepared from H (Heparin) asdescribed. (Inohue, Y., Nagasawa, K. Selective N-desuljation of heparinwith dimethylsulphoxide containing water or methanol. Carbohydr. Res.1976, 46, 87-95.) 50% N-acetyl-heparin (⁵⁰NA-H,) was prepared bysolvolytic partial N-desulfation of H in DMSO/MeOH (9/1, v/v) (Nagasawa,K.; Inoue, Y.; Kamata. T. Solvolytic desuljation ofglycosaminoglycuronan sUljates with dimethyl sulphoxide containing wateror methanol. Carbohydr. Res. 1977, 58, 47-55.) for 120 min at 20° C.,followed by N-acetylation with acetic anhydride in alkaline medium.(Inohue. Y., Nagasawa, K. Selective N-desuljation of heparin withdimethylsulphoxide containing water or methanol. Carbohydr. Res. 1976,46, 87-95.) The N-acetylation degree was determined by integration of¹³C NMR signals at 60 and 55 ppm corresponding to the C-2 of GlcNSO₃ andGlcNAc, respectively. The glycol-split derivative of ⁵⁰NA-H(⁵⁰NA-H-²⁵gs,) ST1518 (N-Ac), was obtained from ⁵⁰NA-H (1 g) dissolvedin 50 mL of 0.1M NaIO₄. The solution was stirred at 4° C. for 16 h inthe dark. The reaction was stopped adding 5 mL ethyleneglycol and thesolution was dialyzed. Solid sodium borohydride (500 mg) was added tothe retentate solution under stirring in several portions. After 2-3 hthe pH was adjusted to 3 with 0.1M HCl, and the solution was neutralizedwith 0.1M NaOH. After dialysis, ⁵⁰NA-H-²⁵gs was recovered in a 46% yieldby freeze drying.

Example 6

LMW-derivatives of H⁴⁶gs ST2184 (LMW-denvative of Sf1514) were preparedby nitrous acid depolymerization. A solution of polysaccharide (4 g)were dissolved in 65 mL H₂O was cooled at 4° C. then added of 75 mg ofNaN0₂ and the pH was adjusted to 2 with 0.1 M HCl. The solution wasstirred at 4° C. for 20 min and then the pH was brought to 7. SolidNaBH₄ (1 g) was added in several portions under stirring. After 2-3 hthe pH was adjusted to 4 with 0.1 M HCl and the solution was neutralizedwith 0.1 M NaOH. The product, obtained by precipitation with 3 volumesof ethanol, was dissolved in water and recovered by freeze-drying. Theextent of glycol-splitting percentage expressed as glycol split residuesreferred to total uronic acids was evaluated by integration of the ¹³CNMR signals at 106.5 ppm and 102 ppm corresponding to C 1 of the splituronic residues and 2-O-sulfated iduronic residues, respectively. Thedepolymerization degree (DP) was evaluated by integration of the ¹³C NMRsignals at 98-107 ppm and 82, 85 and 87 ppm, corresponding to total C1and C2, C3 and C5 of the anhydro-mannitol unit, respectively.

To evaluate the effect of substituents at the level of glycol splitresidues, the 74% glycol-split heparin derivative Odesulfated heparin ingalacturonic form H-⁴²GalA was converted into its glycine and taurinederivatives: (H-⁷⁴gs/G, and H-⁷⁴gs/T).

Example 7 Glycine (H-74gs/G) ST1829, Taurine (H-74gs/T) ST1828 andAmmonium ST1919 Derivatives of Glycol-Split Heparins

These derivatives were obtained by reductive amination (Hoffman. J.;Larm. O.; Scholander. E. A new method for covalent coupling of heparinand other glycosaminoglycans to substances containing primary aminogroups. Carbohydr. Res. 1983. 11 7. 328-331.) of polydialdehydesobtained by periodate oxidation of GalA analogs. All the derivativeswere prepared from the GalA containing intermediate also used forpreparation of H-⁷⁴gs. To prepare the glycine derivative H-⁷⁴gs/G,H-⁴²GalA (300 mg) was dissolved in 7.5 mL of H₂O, then added of 7.5 mL0.2M NaIO₄ solution. After stirring at 4° C. for 16 h in the dark, thereaction was stopped adding 1 mL ethyleneglycol and the solution wasdialyzed for 16 h. The solution volume (about 100 mL) was reduced to 12mL under vacuum, then glycine (1.6 g) was added under stirring inseveral portions. After 1 h solid NaBH₄ (200 mg) was added in severalportions under stirring and the pH was adjusted to 6 with 0.1M HCl.After 3 h the pH was adjusted to 4 with 0.1M HCl, and the solution wasneutralized with 0.1M NaOH. After desalting (which removed alsounreacted glycine), the final product ST1829 was recovered in a 85%yield by freeze drying. Typical ¹³C NMR signals: 52 ppm (NHCH₂CO₂):106.5 ppm (C1 of glycol-split uronic acid residues). The extent ofglycol-splitting (74%) was evaluated as described before forglycol-split heparins. The glycine molar substitution G % (48%, asreferred to total split uronic acids) was evaluated from the area of ¹³CNMR signals at 52 ppm (A) and 106.5 ppm (B), corresponding to CH2glycine signals and split uronic residues, respectively, as follows: G%=A/B×100.

The amine derivative H-⁷⁴gS/A (ST1919) was prepared following the sameprocedure described for H-74gs/G, using NH₄OH instead of glycine (yield:57%).

The compounds of Examples 5-7 are summarized by the following formula:

In the FGF2-mediated cell-cell adhesion test, FGF2 causes cell-cellattachment by linking FGFRs carried on FGFR1-overexpressingHSPG-deficient CHO mutants to HSPGs expressed by neighboring wildtypeCHO-K1 cells. 25 In this assay the glycine derivative ST1829 (H-74gs/G)prevent the formation of the FGFR/FGF2/HSPG ternarycomplex with apotency (ID50=10-30 ng/mL) Similar or better than that shown by heparin(ID50=100 ng/mL).

TABLE 11 Average Molecular Weight (Mw and pd)¹, Sulfation Degree (DS)²,Inhibition of FGF2-mediated Cell-Cell Adhesion³ and Endothelial Cellproliferation,³ and Antiangiogenic Activity in CAM Assay^(4,5) forHeparin and Heparin Derivatives.^(a) CAM CAM Cell-Cell Adh. Cell Prolif.CAM vs FGF-2 vs VEGF⁵ Mw (Pd) DS (ID₅₀, ng/mL) (ID₅₀, ng/mL) (%inhibition)^(b) (% inhibition)^(b) (% inhibition)^(c) Heparin (H) 15 800(1.1) 2.2 100 2 000  0 2-O-desulfated heparins H-⁴²GalA 13 200 (1.2) 1.8300 1 000 nd (Starting Material of H⁷⁴-GS) Glycol-split heparins H-⁴⁶gs11 900 (1.2) 1.7 100   300 80 70 50 ST1514 H-⁷⁴gs 12 800 (1.3) 1.5  10  100 60 ST1827 LMW-H-⁵²gs  5 800 (1.4) 1.7  30   300 70 70 ST2184vLMW-H-⁵²gs  3 000 (2.2) 1.7  30   300 90 ST2010 Derivatives ofglycol-split heparins H-⁷⁴gs/G ~12.000 nd nd  10 3 000 80 ST1829H-⁷⁴gs/T ~.12.000  nd nd 300 1 000 70 ST1828 N-acetylated heparins¹⁰⁰NA-H 11 300 (1.3) 1.5 30 000   >100 000    0 55 40 ST1511 ⁵⁰NA-H 10900 (1.3) 1.8 3 000   1 000  0 ST1512 ⁵⁰NA-H-²⁵gs 12 100 (1.3) 1.7 30030 000  30 30 30 ST1318 ^(a)Percent conversions are indicated assuperscripts on symbols of products; ^(b)100 μg/embryo; nd.; ^(c)VEGF500 ng/embryo: not determined ¹Casu, B.; Gennaro, U. A simpleconductimetric method for determining the sulfate and carboxylate groupsin heparins and chondroitin sulfates. Carbohydr. Res. 1975, 39, 168-176.²Keary, C M. Charaterization of Methocel cellulose ethers by aqueous SECwith multiple detectors. Carbohydr. Polymers 2001, 45, 293-303. ³Leali,D.; Belleri, M; Urbinati, C; Coltrini, D.; Oreste, P.; wppelti, G.;Ribatti, D.; Rusnati, M.; Presta, M. Fibroblast growth factor-2antagonist activity and angiostatic capacity of sulfated Escherichiacoli K5 polysaccharide derivatives. J. Biol. Chem. 2001, 276,37900-37908. ⁴Ribatti, D.; Goulandris, A., Bastaki, M., Vacca, A.,lurlaro, M.; ROllcali, L; Presta, M. New model of angiogenesis andalltiallgiogellesis ill the chick embryo chorioallantoi membralle: thegelatill spollgelchorioallalltoic membrane assay. J. Vase. Res. 1997,34, 455-463. ⁵ Ulldersulfated, low 1Il0leeuiar weight glycol-splitheparin as an antiangiogellie VEGF antagonist. Pisano C, Aulicino C,Vesci L, Casu B, Naggi A, Torri G, Ribatti D, Belleri M, Rusnati M,Presta M. Glycobiology. 2004 Oct. 20.

MeWo melanoma cells (5×105) were inoculated i.d. Animals were treateds.c. twice a day with 25 mg/kg ST2184 for ten days. Mice were sacrificedand the skin areas in contact with the dorsal side of the chamber weretrimmed off. Quantification of the total vascularized area was performedby computer-assisted image analysis.

The following Table 12 shows the results.

TABLE 12 Effect of ST2184 on vascularization of MeWo melanomaxenografted onto nude mice. Daily Dose Vascularized area Treatment(mg/kg) n mm₂ ± SE Vehicle / 10 8.9 ± 0.4 ST2184 50 10 ***5.2 ± 0.5  ***P < 0.001 vs vehicle (Mann-Whitney).

1. A method of inhibiting angiogenesis, comprising administering to asubject a 2-O-desulfated glycosaminoglycan derivative with a iduronicacid desulfation degree not greater than 60% of the total uronic units,which has the following formula (I):

where X and X′ are —CH₂-D group, where D is selected from the groupconsisting of hydroxyl, amino acid, NH₂, HN(CH₂)₂SO₃Na; R and R1, whichare the same or different, are an SO₃ or acetyl residue; n and m, whichare the same or different, vary from 1 to 40; the sum of n+m ranges from6 to 40; the m:n ratio ranges from 10:2 to 1:1, and the symbol

indicates that the units marked m and n are statistically distributedalong the polysaccharide chain and are not necessarily in sequence. 2.The method of claim 1, wherein said derivative is a modified heparinbeing subjected to partial N-desulfation.
 3. A method of inhibitingangiogenesis, comprising administering to a subject a 2-O-desulfatedglycosaminoglycan derivative with a iduronic acid desulfation degree notgreater than 60% of the total uronic units, which has the followingformula:

where R are different and are H or CH₂COO⁻ and where n is
 10. 4. Amethod of inhibiting angiogenesis, comprising administering to a subjecta 2-O-desulfated glycosaminoglycan derivative with a iduronic aciddesulfation degree not greater than 60% of the total uronic units, whichhas the following formula:

where R are different and are H or CH₂CH₂SO₃ ⁻ and n is
 10. 5. A methodof treating a pathological state due to abnormal angiogenesis,comprising administering to a subject a 2-O-desulfated glycosaminoglycanderivative with a iduronic acid desulfation degree not greater than 60%of the total uronic units, which has the following formula (I):

where X and X′ are —CH₂-D group, where D is selected from the groupconsisting of hydroxyl, amino acid, NH₂, HN(CH₂)₂SO₃Na; R and R1, whichare the same or different, are an SO₃ or acetyl residue; n and m, whichare the same or different, vary from 1 to 40; the sum of n+m ranges from6 to 40; the m:n ratio ranges from 10:2 to 1:1, and the symbol

indicates that the units marked m and n are statistically distributedalong the polysaccharide chain and are not necessarily in sequence. 6.The method of claim 5, wherein said derivative is a modified heparinbeing subjected to partial N-desulfation.
 7. A method of treating apathological state due to abnormal angiogenesis comprising administeringto a subject a 2-O-desulfated glycosaminoglycan derivative with aiduronic acid desulfation degree not greater than 60% of the totaluronic units, which has the following formula:

where R are different and are H or CH₂COO⁻ and where n is
 10. 8. Amethod of treating a pathological state due to abnormal angiogenesis,comprising administering to a subject a 2-O-desulfated glycosaminoglycanderivative with a iduronic acid desulfation degree not greater than 60%of the total uronic units, which has the following formula:

where R are different and are H or CH₂CH₂SO₃ ⁻ and n is
 10. 9. Themethod of claim 5, wherein the abnormal angiogenesis is a tumor a tumorassociated with metastases, tumor metastases, diabetic retinopathy,retrolenticular fibroplasias, psoriasis or restenosis after angioplastyor after coronary bypass.
 10. The method of claim 7, wherein theabnormal angiogenesis is a tumor a tumor associated with metastases,tumor metastases, diabetic retinopathy, retrolenticular fibroplasias,psoriasis or restenosis after angioplasty or after coronary bypass. 11.The method of claim 8, wherein the abnormal angiogenesis is a tumor atumor associated with metastases, tumor metastases, diabeticretinopathy, retrolenticular fibroplasias, psoriasis or restenosis afterangioplasty or after coronary bypass.